Biosensor for detecting multiple epitopes on a target

ABSTRACT

The present invention encompasses a method for detecting a target comprising a repeating epitope.

CROSS REFERENCE TO RELATED APPLICATIONS

This application in a continuation of U.S. application Ser. No.13/133,198, filed Oct. 18, 2011, now U.S. Pat. No. 8,993,245, issuedMar. 31, 2015, which is a U.S. National Application of PCT Applicationnumber PCT/US2009/065142, filed Nov. 19, 2009, which claims the priorityof U.S. Provisional Application No. 61/116,875, filed Nov. 21, 2008,each of which is hereby incorporated by reference in its entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under R41 GM079891awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Pathogenic bacteria are responsible for over fifty infectious diseases.Methodologies allowing detection of the pathogens in clinical, food,water and environmental samples are an important component of infectiousdisease diagnosis, treatment and prevention. Currently, pathogendetection involves traditional methods based on cell culture and colonycounting, antigen detection methods, PCR-based methods, and variousbiosensors. Each of these methods has its own strengths and weaknesses.Traditional methods are robust and sensitive, but very slow. Antigen andPCR-based methods are much faster but are technically demanding and inthe case of PCR-based methods, prone to false-positives. Biosensorsoffer the promise of much shorter detection times but require moredevelopment before they become a real alternative. There is clearly aneed for new detection methodologies that would overcome the limitationsof currently existing technologies.

SUMMARY OF THE INVENTION

One aspect of the present invention encompasses a method for detecting atarget comprising at least one repeating epitope. The method comprisescontacting a sample comprising the target with a molecular biosensor.The biosensor comprises:R²⁴—R²⁵—R²⁶—R²⁷,R²⁸—R²⁹—R³⁰—R³¹, andO

-   -   wherein:        -   R²⁴ is a peptide epitope binding agent that binds to a            repeating epitope on a target antibody;        -   R²⁵ is a flexible linker attaching R²⁴ to R²⁶;        -   R²⁶ and R³⁰ are a pair of nucleotide sequences that are not            complementary to each other, but are complementary to two            distinct regions on O;        -   R²⁷ and R³¹ together comprise a detection means such that            when R²⁶ and R³⁰ associate with O a detectable signal is            produced;        -   R²⁸ is a peptide epitope binding agent that binds to the            same repeating epitope on the target antibody as R²⁴;        -   R²⁹ is a flexible linker attaching R²⁸ to R³⁰; and        -   O is a nucleotide sequence comprising a first region that is            complementary to R²⁶, and a second region that is            complementary to R³⁰, and        -   detecting the signal produced by the association of R²⁶ with            O and R³⁰ with O, wherein the signal indicates the presence            of the target antibody.

Other aspects and iterations of the invention are described morethoroughly below.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one photograph executed in color.Copies of this patent application publication with color photographswill be provided by the Office upon request and payment of the necessaryfee.

FIG. 1A depicts the design of a molecular biosensor. ANTI1 and ANTI2depict antibodies labeled with signaling oligonucleotides. T correspondsto the target protein. (FIG. 1B) Proof-of-principle for molecularbiosensors. Black line: emission spectrum of 20 nM ANTI1 labeled withfluorescein. Red line: Emission spectrum of a mixture of 20 nM ANTI1 and25 nM ANTI2 labeled with Cy5. Blue line: 20 nM ANTI1 and 25 nM ANTI2 inthe presence of 20 nM human cardiac troponin I. Excitation was at 490nm. Inset: FRET signals for each sample (emission at 670 nm with theexcitation at 490 nm). (FIG. 1C) Cardiac troponin sensor response at lowconcentrations of the protein.

FIG. 2 depicts the design of antibody-based sensor for detectingpathogens.

FIG. 3A depicts the mechanism of preferential binding of the antibodieslabeled with complementary signaling oligonucleotides next to eachother. (FIG. 3B) Proof-of-principle experiment demonstrating feasibilityof the sensor design illustrated in FIG. 2. False-color fluorescenceimages of the wells of the 96-well microplate containing equimolarmixture of labeled antibodies incubated with indicated amounts of targetcells (O157:H7) or negative control cells (K12) are shown. Top panel isthe image of the emission at 520 nm (excited at 488 nm) and the bottompanel is the image of emission at 670 nm (excited at 488 nm).

FIG. 4 depicts FRET signal produced by the 20 nM/25 nM mixture ofantibodies labeled with fluorescein and Cy5-modified complementarysignaling oligonucleotides at indicated amounts of cells (in 20 mlassay, 384-well microplate). Averages and standard deviations of 3independent measurements are shown. Black symbols: E. coli O157:H7; bluesymbols: E. coli K12.

FIG. 5 depicts fluorescence confocal microscope images of the E. coliO157:H7 cells incubated with the antibody labeled withfluorescein-modified signaling oligonucleotide (donor only) or with themixture of antibody labeled with fluorescein and Cy5-modifiedcomplementary oligonucleotides (donor/acceptor). Fluorescein image wastaken with excitation at 488 nm and emission at 520 nm. FRET image wastaken with excitation at 488 nm and emission at 670 nm.

FIG. 6 depicts the kinetics of the biosensor response. Indicated amountsof E. coli O157:H7 cells were added to 20 nM mixture of antibody labeledwith fluorescein and Cy5-modified complementary oligonucleotides andFRET signal (emission at 670 nm with excitation at 488 nm) was monitoredover time.

FIG. 7 depicts the increased sensitivity of target cell detection atlower antibody concentration. Black symbols: 10/12 nM nM antibodymixture; blue symbols: 5/6.2 nM nM antibody mixture.

FIG. 8 depicts a sample concentration step that can be used to enhancedetectabilty of low amounts of the bacteria. Red bars: 3000 cells/mlsample; blue bars: 300,000 cells/ml sample.

FIG. 9 depicts a biosensor for Salmonella typhimurium. FRET signalproduced by the 20 nM/25 nM mixture of antibodies labeled withfluorescein and Cy5-modified complementary signaling oligonucleotides atindicated amounts of cells (in 20 μl assay, 384-well microplate).Averages and standard deviations of 3 independent measurements areshown. Black symbols: Salmonella typhimurium; cyan symbols: E. coli K12;blue symbols: E. coli O157:H7.

FIG. 10 depicts the kinetics of a Salmonella typhimurium biosensorresponse. Indicated amounts of Salmonella cells were added to 20 nM/25nM mixture of antibody labeled with fluorescein and Cy5-modifiedcomplementary oligonucleotides and FRET signal (emission at 670 nm withexcitation at 488 nm) was monitored over time.

FIG. 11 depicts the homogenous restriction-enzyme based signalamplification methodology compatible with homogenous nature of molecularbiosensors. S1 and S2 correspond to the antibodies recognizing thetarget labeled with short oligonucleotides complementary to the regionof S3 flanking the restriction enzyme cleavage site in S3.

FIG. 12 depicts a design of the solid-surface variant of the sensor fordetecting bacteria. Oligonucleotide containing multiple copies of asequence complementary to the signaling oligonucleotide used forlabeling the antibody is immobilized on the solid surface.

FIG. 13 depicts a proof-of-principle validation of the designillustrated in FIG. 12. Various amounts of E. coli 0157:H7 cells wereincubated in the presence of 20 nM antibody conjugated tofluorescein-modified signaling oligonucleotide in microplate wellscontaining immobilized complementary oligonucleotide. After washing thewells with the buffer the fluorescence remaining in the wells were readon a plate reader. Inset: Specificity of the solid-surface based sensor.No signal in the presence of E. coli K12 was detected (red symbols).

FIG. 14 depicts a proof-of-principle for a TIRF-based solid-surfacebased biosensor for bacteria. Oligonucleotide containing multiple copiesof a sequence complementary to the signaling oligonucleotide used forlabeling the antibody was immobilized on the surface of a quartz slide.TIRF-induced fluorescence originating from the surface of the slide wasmonitored. Only a small signal was observed upon injecting the samplecontaining only labeled antibody whereas large signal was observed inthe presence of target bacteria.

FIG. 15A-B depicts two standard curves for a ferritin assay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of using a molecularbiosensor. In particular, the method comprises detecting targetscomprising repeating epitopes. The method typically involvestarget-molecule induced co-association of two epitope-binding agentsthat each recognize the same repeating epitope on the target. Theepitope-binding agents each comprise complementary signalingoligonucleotides that are labeled with detection means and are attachedto the epitope binding agents through a flexible linker. Co-associationof the two epitope-binding agents with the target results in bringingthe two signaling oligonucleotides into proximity such that a detectablesignal is produced. Advantageously, the molecular biosensors provide arapid homogeneous means to detect a variety of targets comprisingrepeating epitopes, including but not limited to proteins,carbohydrates, lipids, and microbial organisms.

I. Molecular Biosensors

One aspect of the invention, accordingly, encompasses a molecularbiosensor. In one embodiment, the molecular biosensor may be monovalentcomprising a single epitope binding agent that binds to an epitope on atarget. A molecular biosensor of the invention, however, is typicallymultivalent. It will be appreciated by a skilled artisan, depending uponthe target, that the molecular biosensor may comprise from about 2 toabout 5 epitope binding agents. Typically, the molecular biosensor maycomprise 2 or 3 epitope binding agents and more typically, may comprise2 epitope binding agents. In one alternative of this embodiment,therefore, the molecular biosensor may be bivalent comprising a firstepitope binding agent that binds to a repeating epitope on a target anda second epitope binding agent that binds to the same repeating epitopeon the target. In another alternative of this embodiment, the molecularbiosensor may be trivalent comprising a first epitope binding agent thatbinds to a repeating epitope on a target, a second epitope binding agentthat binds to the same repeating epitope on a target and a third epitopebinding agent that binds to the same repeating epitope on a target.

(a) Bivalent Molecular Sensors

In one embodiment of the invention, the molecular biosensor may bebivalent. In a typical embodiment, the bivalent construct will comprisea first epitope binding agent that binds to a repeating epitope on atarget, a first linker, a first signaling oligo, a first detectionmeans, a second epitope binding agent that binds to the same repeatingepitope on the target, a second linker, a second signaling oligo, and asecond detection means.

In one preferred embodiment, the molecular biosensor comprises twonucleic acid constructs, which together have formula (I):R¹—R²—R³—R⁴; andR⁵—R⁶—R⁷—R⁸;  (I)

-   -   wherein:        -   R¹ is an epitope binding agent that binds to a repeating            epitope on a target;        -   R² is a flexible linker attaching R¹ to R³;        -   R³ and R⁷ are a pair of complementary nucleotide sequences            having a free energy for association from about 5.5            kcal/mole to about 8.0 kcal/mole at a temperature from about            21° C. to about 40° C. and at a salt concentration from            about 1 mM to about 100 mM;        -   R⁴ and R⁸ together comprise a detection means such that when            R³ and R⁷ associate a detectable signal is produced;        -   R⁵ is an epitope binding agent that binds to the same            repeating epitope on the target as R¹; and        -   R⁶ is a flexible linker attaching R⁵ to R⁷.

As will be appreciated by those of skill in the art, the choice ofepitope binding agents, R¹ and R⁵, in molecular biosensors havingformula (I) can and will vary depending upon the particular target. Byway of example, when the target is a protein, R¹ and R⁵ may be anaptamer, or antibody. By way of further example, when R¹ and R⁵ aredouble stranded nucleic acid the target is typically a macromoleculethat binds to DNA or a DNA binding protein. In general, suitable choicesfor R¹ and R⁵ will include two agents that each recognize a repeatingepitope on the same target. In certain embodiments, however, it is alsoenvisioned that R¹ and R⁵ may recognize repeating epitopes on differenttargets. Non-limiting examples of suitable epitope binding agents,depending upon the target, include agents selected from the groupconsisting of an aptamer, an antibody, an antibody fragment, adouble-stranded DNA sequence, a ligand, a ligand fragment, a receptor, areceptor fragment, a polypeptide, a peptide, a coenzyme, a coregulator,an allosteric molecule, and an ion. In an exemplary embodiment, R¹ andR⁵ are each antibodies selected from the group consisting of polyclonalantibodies, ascites, Fab fragments, Fab′ fragments, monoclonalantibodies, chimeric antibodies, and humanized antibodies. In apreferred embodiment, R¹ and R⁵ are each monoclonal antibodies. Inanother embodiment, R¹ and R⁵ are each aptamers. In an additionalembodiment, R¹ and R⁵ are each double stranded DNA. In a furtherembodiment, R¹ is a double stranded nucleic acid and R⁵ is an aptamer.In an additional embodiment, R¹ is an antibody and R⁵ is an aptamer. Inan additional embodiment, R¹ is an antibody and R⁵ is a double strandedDNA. In embodiments where R¹ and R⁵ comprise the same epitope bindingagent, the bivalent biosensor may be considered a monovalent biosensor.

In an additional embodiment for molecular biosensors having formula (I),exemplary linkers, R² and R⁶, will functionally keep R³ and R⁷ in closeproximity such that when R¹ and R⁵ each bind to the target, R³ and R⁷associate in a manner such that a detectable signal is produced by thedetection means, R⁴ and R⁸. R² and R⁶ may each be a nucleotide sequencefrom about 10 to about 100 nucleotides in length. In one embodiment, R²and R⁶ are from 10 to about 25 nucleotides in length. In anotherembodiment, R² and R⁶ are from about 25 to about 50 nucleotides inlength. In a further embodiment, R² and R⁶ are from about 50 to about 75nucleotides in length. In yet another embodiment, R² and R⁶ are fromabout 75 to about 100 nucleotides in length. In each embodiment, thenucleotides comprising the linkers may be any of the nucleotide bases inDNA or RNA (A, C, T, G in the case of DNA, or A, C, U, G in the case ofRNA). In one embodiment R² and R⁶ are comprised of DNA bases. In anotherembodiment, R² and R⁶ are comprised of RNA bases. In yet anotherembodiment, R² and R⁶ are comprised of modified nucleic acid bases, suchas modified DNA bases or modified RNA bases. Examples of suitablemodified DNA or RNA bases include 2′-fluoro nucleotides, 2′-aminonucleotides, 5′-aminoallyl-2′-fluoro nucleotides and phosphorothioatenucleotides. Alternatively, R² and R⁶ may be a polymer of bifunctionalchemical linkers. In one embodiment the bifunctional chemical linker isheterobifunctional. Suitable heterobifunctional chemical linkers includesulfoSMCC(Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), andIc-SPDP(N-Succinimidyl-6-(3′-(2-PyridylDithio)-Propionamido)-hexanoate).In another embodiment the bifunctional chemical linker ishomobifunctional. Suitable homobifunctional linkers includedisuccinimidyl suberate, disuccinimidyl glutarate, and disuccinimidyltartrate. Additional suitable linkers are illustrated in the Examples,such as the phosphoramidate form of Spacer 18 comprised of polyethyleneglycol. In one embodiment, R² and R⁶ are from 0 to about 500 angstromsin length. In another embodiment, R² and R⁶ are from about 20 to about400 angstroms in length. In yet another embodiment, R² and R⁶ are fromabout 50 to about 250 angstroms in length.

In a further embodiment for molecular biosensors having formula (I), R³and R⁷ are complementary nucleotide sequences having a length such thatthey preferably do not associate unless R¹ and R⁵ bind to separaterepeating epitopes on the target. When R¹ and R⁵ bind to separateepitopes of the target, R³ and R⁷ are brought to relative proximityresulting in an increase in their local concentration, which drives theassociation of R³ and R⁷. R³ and R⁷ may be from about 2 to about 20nucleotides in length. In another embodiment, R³ and R⁷ are from about 4to about 15 nucleotides in length. In an exemplary embodiment, R³ and R⁷are from about 5 to about 7 nucleotides in length. In one embodiment, R³and R⁷ have a free energy for association from about 5.5 kcal/mole toabout 8.0 kcal/mole as measured in the selection buffer conditions,defined below. In another embodiment, R³ and R⁷ have a free energy forassociation from about 6.0 kcal/mole to about 8.0 kcal/mole as measuredin the selection buffer conditions defined below. In yet anotherembodiment, R³ and R⁷ have a free energy for association from about 7.0kcal/mole to 8.0 kcal/mole in the selection buffer conditions. In apreferred embodiment, R³ and R⁷ have a free energy for association of7.5 kcal/mole in the selection buffer conditions described below.Preferably, in each embodiment R³ and R⁷ are not complementary to R¹ andR⁵.

In a typical embodiment for molecular biosensors having formula (I), R⁴and R⁵ may together comprise several suitable detection means such thatwhen R³ and R⁷ associate, a detectable signal is produced. Exemplarydetections means suitable for use in the molecular biosensors mayinclude FRET, fluorescence cross-correlation spectroscopy, flourescencequenching, fluorescence polarization, flow cytometry, scintillationproximity, luminescence resonance energy transfer, direct quenching,ground-state complex formation, chemiluminescence energy transfer,bioluminescence resonance energy transfer, excimer formation,colorimetric substrates detection, phosphorescence, electro-chemicalchanges, and redox potential changes.

In a further embodiment, the molecular biosensor will have formula (I)wherein:

-   -   R¹ is an epitope binding agent that binds to a repeating epitope        on a target and is selected from the group consisting of an        aptamer, an antibody, and double stranded nucleic acid;    -   R² is a flexible linker attaching R¹ to R³ by formation of a        covalent bond with each of R¹ and R³, wherein R² comprises a        bifunctional chemical cross linker and is from 0 to 500        angstroms in length;    -   R³ and R⁷ are a pair of complementary nucleotide sequences from        about 4 to about 15 nucleotides in length and having a free        energy for association from about 5.5 kcal/mole to about 8.0        kcal/mole at a temperature from about 21° C. to about 40° C. and        at a salt concentration from about 1 mM to about 100 mM;    -   R⁴ and R⁵ together comprise a detection means selected from the        group consisting of FRET, fluorescence cross-correlation        spectroscopy, flourescence quenching, fluorescence polarization,        flow cytometry, scintillation proximity, luminescence resonance        energy transfer, direct quenching, ground-state complex        formation, chemiluminescence energy transfer, bioluminescence        resonance energy transfer, excimer formation, colorimetric        substrates detection, phosphorescence, electro-chemical changes,        and redox potential changes;    -   R⁵ is an epitope binding agent that binds to the same repeating        epitope on the target as R¹, and is selected from the group        consisting of an aptamer, an antibody, and double stranded        nucleic acid; and    -   R⁶ is a flexible linker attaching R⁵ to R⁷ by formation of a        covalent bond with each of R⁵ and R⁷, wherein R⁶ comprises a        bifunctional chemical cross linker and is from 0 to 500        angstroms in length.

Yet another embodiment of the invention encompasses a molecularbiosensor having formula (I) wherein:

-   -   R¹ is an antibody that binds to a repeating epitope on a target;    -   R² is a flexible linker attaching R¹ to R³;    -   R³ and R⁷ are a pair of complementary nucleotide sequences        having a free energy for association from about 5.5 kcal/mole to        about 8.0 kcal/mole at a temperature from about 21° C. to about        40° C. and at a salt concentration from about 1 mM to about 100        mM;    -   R⁴ and R⁸ together comprise a detection means such that when R³        and R⁷ associate a detectable signal is produced;    -   R⁵ is an antibody that binds to the same repeating epitope on        the target as R¹; and    -   R⁶ is a flexible linker attaching R⁵ to R⁷.

A further embodiment of the invention encompasses a molecular biosensorhaving formula (I) wherein:

-   -   R¹ is an antibody that binds to a repeating epitope on a target;    -   R² is a flexible linker attaching R¹ to R³ by formation of a        covalent bond with each of R¹ and R³, wherein R² comprises a        bifunctional chemical cross linker and is from 0 to 500        angstroms in length;    -   R³ and R⁷ are a pair of complementary nucleotide sequence from        about 4 to about 15 nucleotides in length and having a free        energy for association from about 5.5 kcal/mole to about 8.0        kcal/mole at a temperature from about 21° C. to about 40° C. and        at a salt concentration from about 1 mM to about 100 mM;    -   R⁴ and R⁸ together comprise a detection means selected from the        group consisting of FRET, fluorescence cross-correlation        spectroscopy, flourescence quenching, fluorescence polarization,        flow cytometry, scintillation proximity, luminescence resonance        energy transfer, direct quenching, ground-state complex        formation, chemiluminescence energy transfer, bioluminescence        resonance energy transfer, excimer formation, colorimetric        substrates detection, phosphorescence, electro-chemical changes,        and redox potential changes;    -   R⁵ is an antibody that binds to the same repeating epitope on        the target; and    -   R⁶ is a flexible linker attaching R⁵ to R⁷ by formation of a        covalent bond with each of R⁵ and R⁷, wherein R⁶ comprises a        bifunctional chemical cross linker and is from 0 to 500        angstroms in length.        (b) Trivalent Molecular Sensors

In an additional alternative embodiment, the molecular biosensor may betrivalent. In a typical embodiment, the trivalent construct may comprisea first epitope binding agent that binds to a repeating epitope on atarget, a first linker, a first signaling oligo, a first detectionmeans, a second epitope binding agent that binds to the same repeatingepitope on the target, a second linker, a second signaling oligo, asecond detection means, a third epitope binding agent that binds to thesame repeating epitope on a target, a third linker, a third signalingoligo, and a third detection means.

In one preferred embodiment, the molecular biosensor comprises threenucleic acid constructs, which together have formula (II):R¹⁵—R¹⁴—R¹³—R⁹—R¹⁰—R¹¹—R¹²;R¹⁶—R¹⁷—R¹⁸—R¹⁹; andR²⁰—R²¹—R²²—R²³  (II)

-   -   wherein:        -   R⁹ is an epitope binding agent that binds to a repeating            epitope on a target;        -   R¹⁰ is a flexible linker attaching R⁹ to R¹¹;        -   R¹¹ and R²² are a first pair of complementary nucleotide            sequences having a free energy for association from about            5.5 kcal/mole to about 8.0 kcal/mole at a temperature from            about 21° C. to about 40° C. and at a salt concentration            from about 1 mM to about 100 mM;        -   R¹² and R²³ together comprise a detection means such that            when R¹¹ and R²² associate a detectable signal is produced;        -   R¹³ is a flexible linker attaching R⁹ to R¹⁴;        -   R¹⁴ and R¹⁸ are a second pair of complementary nucleotide            sequences having a free energy for association from about            5.5 kcal/mole to about 8.0 kcal/mole at a temperature from            about 21° C. to about 40° C. and at a salt concentration            from about 1 mM to about 100 mM;        -   R¹⁵ and R¹⁹ together comprise a detection means such that            when R¹⁴ and R¹⁸ associate a detectable signal is produced;        -   R¹⁶ is an epitope binding agent that binds to the same            repeating epitope on a target as R⁹;        -   R¹⁷ is a flexible linker attaching R¹⁶ to R¹⁸;        -   R²⁰ is an epitope binding agent that binds to the same            repeating epitope on a target as R⁹ and R¹⁶; and        -   R²¹ is a flexible linker attaching R²⁰ to R²².

The choice of epitope binding agents, R⁹, R¹⁶ and R²⁰, in molecularbiosensors having formula (II) can and will vary depending upon theparticular target. Generally speaking, suitable choices for R⁹, R¹⁶ andR²⁰ will include three agents that each recognize the same repeatingepitope on the same target or on different targets. Non-limitingexamples of suitable epitope binding agents, depending upon thetarget(s), include agents selected from the group consisting of anaptamer, an antibody, an antibody fragment, a double-stranded DNAsequence, a ligand, a ligand fragment, a receptor, a receptor fragment,a polypeptide, a peptide, a coenzyme, a coregulator, an allostericmolecule, and an ion. In an exemplary embodiment, R⁹, R¹⁶ and R²⁰ areeach antibodies.

In an additional embodiment for molecular biosensors having formula(II), exemplary linkers, R¹⁰ and R²¹, will functionally keep R¹¹ and R²²in close proximity such that when R⁹ and R²⁰ each bind to the repeatingepitopes on the target(s), R¹¹ and R²² associate in a manner such that adetectable signal is produced by the detection means, R¹² and R²³. Inaddition, exemplary linkers, R¹³ and R¹⁷, will functionally keep R¹⁴ andR¹⁸ in close proximity such that when R⁹ and R¹⁶ each bind to therepeating epitopes on the target(s), R¹⁴ and R¹⁸ associate in a mannersuch that a detectable signal is produced by the detection means, R¹⁵and R¹⁹. In one embodiment, the linkers utilized in molecular biosensorshaving formula (II) may each be a nucleotide sequence from about 10 toabout 100 nucleotides in length. In one embodiment, the linkers are from10 to about 25 nucleotides in length. In another embodiment, the linkersare from about 25 to about 50 nucleotides in length. In a furtherembodiment, the linkers are from about 50 to about 75 nucleotides inlength. In yet another embodiment, the linkers are from about 75 toabout 100 nucleotides in length. In each embodiment, the nucleotidescomprising the linkers may be any of the nucleotide bases in DNA or RNA(A, C, T, G in the case of DNA, or A, C, U, G in the case of RNA). Inone embodiment, the linkers are comprised of DNA bases. In anotherembodiment, the linkers are comprised of RNA bases. In yet anotherembodiment, the linkers are comprised of modified nucleic acid bases,such as modified DNA bases or modified RNA bases. Examples of suitablemodified DNA or RNA bases include 2′-fluoro nucleotides, 2′-aminonucleotides, 5′-aminoallyl-2′-fluoro nucleotides and phosphorothioatenucleotides. Alternatively, the linkers may be a polymer of bifunctionalchemical linkers. In one embodiment the bifunctional chemical linker isheterobifunctional. Suitable heterobifunctional chemical linkers includesulfoSMCC(Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), andIc-SPDP(N-Succinimidyl-6-(3′-(2-PyridylDithio)-Propionamido)-hexanoate).In another embodiment the bifunctional chemical linker ishomobifunctional. Suitable homobifunctional linkers includedisuccinimidyl suberate, disuccinimidyl glutarate, and disuccinimidyltartrate. Additional suitable linkers are illustrated in the Examples,such as the phosphoramidate form of Spacer 18 comprised of polyethyleneglycol. In one embodiment, the linkers are from 0 to about 500 angstromsin length. In another embodiment, the linkers are from about 20 to about400 angstroms in length. In yet another embodiment, the linkers are fromabout 50 to about 250 angstroms in length.

In a further embodiment for molecular biosensors having formula (II),R¹¹ and R²² are complementary nucleotide sequences having a length suchthat they preferably do not associate unless R⁹ and R²⁰ bind to separaterepeating epitopes on the target(s). In addition, R¹⁴ and R¹⁸ arecomplementary nucleotide sequences having a length such that theypreferably do not associate unless R⁹ and R¹⁶ bind to separate repeatingepitopes on the target(s). R¹¹ and R²² and R¹⁴ and R¹⁸ may be from about2 to about 20 nucleotides in length. In another embodiment, R¹¹ and R²²and R¹⁴ and R¹⁸ are from about 4 to about 15 nucleotides in length. Inan exemplary embodiment, R¹¹ and R²² and R¹⁴ and R¹⁸ are from about 5 toabout 7 nucleotides in length. In one embodiment, R¹¹ and R²² and R¹⁴and R¹⁸ have a free energy for association from about 5.5 kcal/mole toabout 8.0 kcal/mole as measured in the selection buffer conditions,defined below. In another embodiment, R¹¹ and R²² and R¹⁴ and R¹⁸ have afree energy for association from about 6.0 kcal/mole to about 8.0kcal/mole as measured in the selection buffer conditions defined below.In yet another embodiment, R¹¹ and R²² and R¹⁴ and R¹⁸ have a freeenergy for association from about 7.0 kcal/mole to 8.0 kcal/mole in theselection buffer conditions. In a preferred embodiment, R¹¹ and R²² andR¹⁴ and R¹⁸ have a free energy for association of 7.5 kcal/mole in theselection buffer conditions described below. Preferably, in eachembodiment R¹¹ and R²² and R¹⁴ and R¹⁸ are not complementary to any ofR⁹, R¹⁶ or R²⁰.

In a typical embodiment for molecular biosensors having formula (II),R¹² and R²³ may together comprise several suitable detection means suchthat when R¹¹ and R²² associate, a detectable signal is produced. Inaddition, R¹⁵ and R¹⁹ may together comprise several suitable detectionmeans such that when R¹⁴ and R¹⁸ associate, a detectable signal isproduced. Exemplary detections means suitable for use in the molecularbiosensors include FRET, fluorescence cross-correlation spectroscopy,flourescence quenching, fluorescence polarization, flow cytometry,scintillation proximity, luminescence resonance energy transfer, directquenching, ground-state complex formation, chemiluminescence energytransfer, bioluminescence resonance energy transfer, excimer formation,colorimetric substrates detection, phosphorescence, electro-chemicalchanges, and redox potential changes.

(c) Three-Component Molecular Biosensors

Another embodiment of the invention comprises three-component molecularbiosensors. These biosensors, in addition to having at least two epitopebinding agent constructs, further comprise an oligonucleotide construct,referred to below as “O.” In certain embodiments, the three-componentmolecular biosensor will comprise an endonuclease restriction site. Inalternative embodiments, the three-component molecular biosensor willnot have an endonuclease restriction site.

i. Biosensors with No Endonuclease Restriction Site

In one embodiment, the three-component biosensor will comprise: (1) afirst epitope binding agent construct that binds to a repeating epitopeon a target, a first linker, a first signaling oligo, and a firstdetection means; (2) a second epitope binding agent construct that bindsto the same repeating epitope on the target, a second linker, a secondsignaling oligo, and a second detection means; and (3) anoligonucleotide construct that comprises a first region that iscomplementary to the first oligo and a second region that iscomplementary to the second oligo. The first signaling oligo and secondsignaling oligo, as such, are not complementary to each other, but arecomplementary to two distinct regions on the oligonucleotide construct.Co-association of the two epitope-binding agent constructs with thetarget results in hybridization of each signaling oligos to theoligonucleotide construct. Binding of the two signaling oligo to theoligonucleotide construct brings them into proximity such that adetectable signal is produced.

In an exemplary embodiment, the three-component molecular biosensorcomprises three nucleic acid constructs, which together have formula(III):R²⁴—R²⁵—R²⁶—R²⁷;R²⁸—R²⁹—R³⁰—R³¹;O  (III)

-   -   wherein:        -   R²⁴ is an epitope-binding agent that binds to repeating            epitope on a target;        -   R²⁵ is a flexible linker attaching R²⁴ to R²⁶;        -   R²⁶ and R³⁰ are a pair of nucleotide sequences that are not            complementary to each other, but are complementary to two            distinct regions on O;        -   R²⁷ and R³¹ together comprise a detection means such that            when R²⁶ and R³⁰ associate a detectable signal is produced;        -   R²⁸ is an epitope-binding agent that binds to the same            epitope on the target as R²⁴;        -   R²⁹ is a flexible linker attaching R²⁸ to R³⁰; and        -   O is a nucleotide sequence comprising a first region that is            complementary to R²⁶, and a second region that is            complementary to R³⁰.

The choice of epitope binding agents, R²⁴ and R²⁸, in molecularbiosensors having formula (III) can and will vary depending upon theparticular target. By way of example, when the target is a protein, R²⁴and R²⁸ may be an aptamer, or antibody. By way of further example, whenR²⁴ and R²⁸ are double stranded nucleic acid the target is typically amacromolecule that binds to DNA or a DNA binding protein. In general,suitable choices for R²⁴ and R²⁸ will include two agents that eachrecognize the same repeating epitope on the same target. In certainembodiments, however, it is also envisioned that R²⁴ and R²⁸ mayrecognize distinct epitopes on different targets. Non-limiting examplesof suitable epitope binding agents, depending upon the target, includeagents selected from the group consisting of an aptamer, an antibody, anantibody fragment, a double-stranded DNA sequence, modified nucleicacids, nucleic acid mimics, a ligand, a ligand fragment, a receptor, areceptor fragment, a polypeptide, a peptide, a coenzyme, a coregulator,an allosteric molecule, and an ion. In an exemplary embodiment, R²⁴ andR²⁸ are each antibodies selected from the group consisting of polyclonalantibodies, ascites, Fab fragments, Fab′ fragments, monoclonalantibodies, chimeric antibodies, and humanized antibodies. In anotherembodiment, R²⁴ and R²⁸ are each aptamers. In an alternative embodiment,R²⁴ and R²⁸ are peptides. In a preferred alternative of this embodiment,R²⁴ and R²⁸ are each monoclonal antibodies. In an additional embodiment,R²⁴ and R²⁸ are each double stranded DNA. In a further embodiment, R²⁴is a double stranded nucleic acid and R²⁸ is an aptamer. In anadditional embodiment, R²⁴ is an antibody and R²⁸ is an aptamer. Inanother additional embodiment, R²⁴ is an antibody and R²⁸ is a doublestranded DNA.

In an additional embodiment for molecular biosensors having formula(III), exemplary linkers, R²⁵ and R²⁹ may each be a nucleotide sequencefrom about 10 to about 100 nucleotides in length. In one embodiment, R²⁵and R²⁹ are from 10 to about 25 nucleotides in length. In anotherembodiment, R² and R⁶ are from about 25 to about 50 nucleotides inlength. In a further embodiment, R²⁵ and R²⁹ are from about 50 to about75 nucleotides in length. In yet another embodiment, R²⁵ and R²⁹ arefrom about 75 to about 100 nucleotides in length. In each embodiment,the nucleotides comprising the linkers may be any of the nucleotidebases in DNA or RNA (A, C, T, G in the case of DNA, or A, C, U, G in thecase of RNA). In one embodiment R²⁵ and R²⁹ are comprised of DNA bases.In another embodiment, R²⁵ and R²⁹ are comprised of RNA bases. In yetanother embodiment, R²⁵ and R²⁹ are comprised of modified nucleic acidbases, such as modified DNA bases or modified RNA bases. Modificationsmay occur at, but are not restricted to, the sugar 2′ position, the C-5position of pyrimidines, and the 8-position of purines. Examples ofsuitable modified DNA or RNA bases include 2′-fluoro nucleotides,2′-amino nucleotides, 5′-aminoallyl-2′-fluoro nucleotides andphosphorothioate nucleotides (monothiophosphate and dithiophosphate). Ina further embodiment, R²⁵ and R²⁹ may be nucleotide mimics. Examples ofnucleotide mimics include locked nucleic acids (LNA), peptide nucleicacids (PNA), and phosphorodiamidate morpholino oligomers (PMO).

Alternatively, R²⁵ and R²⁹ may be a polymer of bifunctional chemicallinkers. In one embodiment the bifunctional chemical linker isheterobifunctional. Suitable heterobifunctional chemical linkers includesulfoSMCC(Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), andIc-SPDP(N-Succinimidyl-6-(3′-(2-PyridylDithio)-Propionamido)-hexanoate).In another embodiment the bifunctional chemical linker ishomobifunctional. Suitable homobifunctional linkers includedisuccinimidyl suberate, disuccinimidyl glutarate, and disuccinimidyltartrate. Additional suitable linkers are illustrated in the Examples,such as the phosphoramidate form of Spacer 18 comprised of polyethyleneglycol. In one embodiment, R²⁵ and R²⁹ are from 0 to about 500 angstromsin length. In another embodiment, R²⁵ and R²⁹ are from about 20 to about400 angstroms in length. In yet another embodiment, R²⁵ and R²⁹ are fromabout 50 to about 250 angstroms in length.

In a further embodiment for molecular biosensors having formula (III),R²⁶ and R³⁰ are nucleotide sequences that are not complementary to eachother, but that are complementary to two distinct regions of 0. R²⁶ andR³⁰ may be from about 2 to about 20 nucleotides in length. In anotherembodiment, R²⁶ and R³⁰ are from about 4 to about 15 nucleotides inlength. In an exemplary embodiment, R²⁶ and R³⁰ are from about 5 toabout 7 nucleotides in length. Preferably, in each embodiment R²⁶ andR³⁰ are not complementary to R²⁴ and R²⁸.

In a typical embodiment for molecular biosensors having formula (III),R²⁷ and R³¹ may together comprise several suitable detection means suchthat when R²⁶ and R³⁰ each bind to complementary, distinct regions on O,a detectable signal is produced. Exemplary detections means suitable foruse in the molecular biosensors include fluorescent resonance energytransfer (FRET), lanthamide resonance energy transfer (LRET),fluorescence cross-correlation spectroscopy, flourescence quenching,fluorescence polarization, flow cytometry, scintillation proximity,luminescence resonance energy transfer, direct quenching, ground-statecomplex formation, chemiluminescence energy transfer, bioluminescenceresonance energy transfer, excimer formation, colorimetric substratesdetection, phosphorescence, electro-chemical changes, and redoxpotential changes.

For molecular biosensors having formula (III), O comprises a firstregion that is complementary to R²⁶, and a second region that iscomplementary to R³⁰. O may be from about 8 to about 100 nucleotides inlength. In other embodiments, 0 is from about 10 to about 15 nucleotidesin length, or from about 15 to about 20 nucleotides in length, or fromabout 20 to about 25 nucleotides in length, or from about 25 to about 30nucleotides in length, or from about 30 to about 35 nucleotides inlength, or from about 35 to about 40 nucleotides in length, or fromabout 40 to about 45 nucleotides in length, or from about 45 to about 50nucleotides in length, or from about 50 to about 55 nucleotides inlength, or from about 55 to about 60 nucleotides in length, or fromabout 60 to about 65 nucleotides in length, or from about 65 to about 70nucleotides in length, or from about 70 to about 75 nucleotides inlength, or from about 75 to about 80 nucleotides in length, or fromabout 80 to about 85 nucleotides in length, or from about 85 to about 90nucleotides in length, or from about 90 to about 95 nucleotides inlength, or greater than about 95 nucleotides in length.

In an exemplary embodiment, 0 will comprise formula (IV):R³²—R³³—R³⁴—R³⁵—R³⁶   (IV)

-   -   wherein:        -   R³², R³⁴, and R³⁶ are nucleotide sequences not complementary            to any of R²⁶, R³⁰, R³³, or R³⁵. R³², R³⁴, and R³⁶ may            independently be from about 2 to about 20 nucleotides in            length. In other embodiments, R³², R³⁴, and R³⁶ may            independently be from about 2 to about 4 nucleotides in            length, or from about 4 to about 6 nucleotides in length, or            from about 6 to about 8 nucleotides in length, or from about            8 to about 10 nucleotides in length, or from about 10 to            about 12 nucleotides in length, or from about 12 to about 14            nucleotides in length, or from about 14 to about 16            nucleotides in length, or from about 16 to about 18            nucleotides in length, or from about 18 to about 20            nucleotides in length, or greater than about 20 nucleotides            in length;        -   R³³ is a nucleotide sequence complementary to R²⁶, and        -   R³⁵ is a nucleotide sequence that is complementary to R³⁰.

R³³ and R³⁵ generally have a length such that the free energy ofassociation between R³³ and R²⁶ and R³⁵ and R³⁰ is from about −5 toabout −12 kcal/mole at a temperature from about 21° C. to about 40° C.and at a salt concentration from about 1 mM to about 100 mM. In otherembodiments, the free energy of association between R³³ and R²⁶ and R³⁵and R³⁰ is about −5 kcal/mole, about −6 kcal/mole, about −7 kcal/mole,about −8 kcal/mole, about −9 kcal/mole, about −10 kcal/mole, about −11kcal/mole, or greater than about −12 kcal/mole at a temperature fromabout 21° C. to about 40° C. and at a salt concentration from about 1 mMto about 100 mM. In additional embodiments, R³³ and R³⁵ may range fromabout 4 to about 20 nucleotides in length. In other embodiments, R³³ andR³⁵ may about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, about 16, about 17,about 18, about 19, or greater than about 10 nucleotides in length.

ii. Biosensors with an Endonuclease Restriction Site

In an alternative embodiment, the three-component biosensor willcomprise: (1) a first epitope binding agent construct that binds to arepeating epitope on a target, a first linker, and a first signalingoligo; (2) a second epitope binding agent construct that binds to thesame repeating epitope on the target, a second linker, a secondsignaling oligo and (3) an oligonucleotide construct that comprises afirst region that is complementary to the first oligo, a second regionthat is complementary to the second oligo, two flexible linkers, anendonuclease restriction site overlapping the first and the secondregions complementary to the first and the second oligos, and a pair ofcomplementary nucleotides with detection means. The first signalingoligo and second signaling oligo are not complementary to each other,but are complementary to two distinct regions on the oligonucleotideconstruct. When the oligonucleotide construct is intact, thecomplementary nucleotides are annealed and produce a detectable signal.Co-association of the two epitope-binding agent constructs with thetarget results in hybridization of each signaling oligo to theoligonucleotide construct. The signaling oligos hybridize to twodistinct locations on the oligonucleotide construct such that adouble-stranded DNA molecule containing the restriction site isproduced, with a gap between the signaling oligos located exactly at thesite of endonuclease cleavage in one strand of the double-stranded DNAsubstrate. When a restriction endonuclease is present, accordingly, itwill cleave the oligonucleotide construct only when the target ispresent (i.e., when the signaling oligos are bound to theoligonucleotide construct). Upon this cleavage, the detection meanspresent on the oligonucleotide are separated-resulting in no detectablesignal. Upon dissociation of the cleaved oligonucleotide construct,another oligonucleotide construct may hybridize with the signalingoligos of the two epitope-binding agents co-associated with the targetand the cleavage reaction may be repeated. This cycle of hybridizationand cleavage may be repeated many times resulting in cleavage ofmultiple oligonucleotide constructs per one complex of the twoepitope-binding agents with the target.

In an exemplary alternative of this embodiment, the three-componentmolecular biosensor comprises three nucleic acid constructs, whichtogether have formula (V):R³⁶—R³⁷R³⁸;R³⁹—R⁴⁰—R⁴¹;O  (V)

-   -   wherein:        -   R³⁶ is an epitope-binding agent that binds to a repeating            epitope on a target;        -   R³⁷ is a flexible linker attaching R³⁶ to R³⁸;        -   R³⁸ and R⁴¹ are a pair of nucleotide sequences that are not            complementary to each other, but are complementary to two            distinct regions on O;        -   R³⁹ is an epitope-binding agent that binds to the same            repeating epitope on the target;        -   R⁴⁰ is a flexible linker attaching R³⁹ to R⁴¹; and O            comprises:            R⁴²—R⁴³—R⁴⁴—R⁴⁵—R⁴⁶    -   R⁴² is a nucleotide construct comprising an endonuclease        restriction site, a first region that is complementary to R³⁸,        and a second region that is complementary to R⁴¹.    -   R⁴³ is a first flexible linker;    -   R⁴⁴ is a first nucleotide sequence that is complementary to R⁴⁶        attached to a detection means;    -   R⁴⁵ is a second flexible linker;    -   R⁴⁶ is a second nucleotide sequence that is complementary to R⁴⁴        attached to a second detection means; and    -   R⁴³ attaches R⁴² to R⁴⁴ and R⁴⁵ attaches R⁴² to R⁴⁶.

Suitable linkers, epitope binding agents, and detection means forthree-component molecular biosensors having formula (V) are the same asthree component molecular biosensors having formula (III). Suitable,endonuclease restriction sites comprising R⁴² include sites that arerecognized by restriction enzymes that cleave double stranded nucleicacid, but not single stranded nucleic acid. By way of non-limitingexample, these sites may include AccI, AgeI, BamHI, BgI, BgII, BsiWI,BstBI, ClaI, CviQI, DdeI, DpnI, DraI, EagI, EcoRI, EcoRV, FseI, FspI,HaeII, HaeIII, HhaI, Hinc II, HinDIII, HpaI, HpaII, KpnI, KspI, MboI,MfeI, NaeI, NarI, NcoI, NdeI, NheI, NotI, PhoI, PstI, PvuI, PvuII, SacI,SacII, SaII, SbfI, SmaI, SpeI, SphI, StuI, TaqI, TfiI, TllI, XbaI, XhoI,XmaI, XmnI, and Zral. Optionally, R⁴² may comprise nucleotide spacersthat precede or follow one or more of the endonuclease restriction site,the first region that is complementary to R³⁸, and/or the second regionthat is complementary to R⁴¹. Suitable nucleotide spacers, for example,are detailed in formula (IV).

II. Methods of Using the Molecular Biosensors

A further aspect of the invention encompasses the use of the molecularbiosensors of the invention in several applications. In certainembodiments, the molecular biosensors are utilized in methods fordetecting one or more targets. In other embodiments, the molecularbiosensors may be utilized in kits and for therapeutic applications.

Typically, a signal produced by a biosensor of the invention may bedetectable in about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In certainembodiments, 80% of the maximum signal may be produced in about 3, 4, 5,6, 7 or 8 minutes. In other embodiments, the maximum signal may beproduced in about 5, 10, 15, 20, 25, 30, 35, 40, or 45 minutes.

(a) Detection Methods

In one embodiment, the molecular biosensors may be utilized for thedetection of a target. The method generally involves contacting amolecular biosensor of the invention with the target. To detect atarget, the method typically involves target-molecule inducedco-association of two epitope-binding agents (present on the molecularbiosensor of the invention) that each recognize the same repeatingepitope on the target. The epitope-binding agents each comprisecomplementary signaling oligonucleotides that are labeled with detectionmeans and are attached to the epitope binding agents through a flexiblelinker. Co-association of the two epitope-binding agents with the targetresults in bringing the two signaling oligonucleotides into proximitysuch that a detectable signal is produced. Typically, the detectablesignal is produced by any of the detection means known in the art or asdescribed herein.

In one particular embodiment, a method for the detection of a targetthat is a protein or polypeptide is provided. The protein or polypeptidecomprises a repeating epitope. The method generally involves detecting aprotein or polypeptide in a sample comprising the steps of contacting asample with a molecular biosensor of the invention.

In another embodiment, the molecular biosensors may be used to detect atarget that is a macromolecular complex in a sample. In this embodiment,the repeating epitope is preferably on each component of themacromolecular complex, such that when the complex is formed, theepitope-binding agents that recognize the repeating epitope are boughtinto proximity, resulting in the stable interaction of the firstsignaling oligo and the second signaling oligo to produce a detectablesignal, as described above.

(b) Solid Surfaces

Optionally, the invention also encompasses a solid surface having one ormore of the the molecular constructs of the invention attached thereto.Non-limiting examples of suitable surfaces include microtitre plates,test tubes, beads, resins and other polymers, as well as other surfaceseither known in the art or described herein. In one embodiment, thesolid surface comprises a nucleic acid sequence that is complementary toa signaling oligo of one or more molecular constructs, as described inthe examples. Therefore, when the epitope binding agent recognizes thetarget comprising repeating epitopes, the signaling oligo of themolecular construct binds to the immobilized nucleic acid sequence. Thebinding may be detected by any means detailed above or in the examples.

In another embodiment the solid surface utilizes a three-componentbiosensor. In this embodiment, the oligonucleotide construct may beimmobilized on a solid surface. The first epitope binding agent andsecond epitope binding agent are contacted with the surface comprisingimmobilized O and a sample that may comprise a target. In the presenceof target, the first epitope binding agent, second epitope bindingagent, and target bind to immobilized O to form a complex. Severalmethods may be utilized to detect the presence of the complex comprisingtarget. The method may include detecting a probe attached to theepitope-binding agents after washing out the unbound components.Alternatively, several surface specific real-time detection methods maybe employed, including but not limited to surface plasmon resonance(SPR) or total internal reflection fluorescence (TIRF).

The oligonucleotide construct, O, may be immobilized to several types ofsuitable surfaces. The surface may be a material that may be modified tocontain discrete individual sites appropriate for the attachment orassociation of the three-component biosensor and is amenable to at leastone detection method. Non-limiting examples of surface materials includeglass, modified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ,etc.), nylon or nitrocellulose, polysaccharides, nylon, resins, silicaor silica-based materials including silicon and modified silicon,carbon, metals, inorganic glasses and plastics. The size and shape ofthe surface may also vary without departing from the scope of theinvention. A surface may be planar, a surface may be a well, i.e. a 364well plate, or alternatively, a surface may be a bead or a slide.

The oligonucleotide construct, O, may be attached to the surface in awide variety of ways, as will be appreciated by those in the art. O, forexample, may either be synthesized first, with subsequent attachment tothe surface, or may be directly synthesized on the surface. The surfaceand O may be derivatized with chemical functional groups for subsequentattachment of the two. For example, the surface may be derivatized witha chemical functional group including, but not limited to, amino groups,carboxyl groups, oxo groups or thiol groups. Using these functionalgroups, the O may be attached using functional groups either directly orindirectly using linkers. Alternatively, O may also be attached to thesurface non-covalently. For example, a biotinylated O can be prepared,which may bind to surfaces covalently coated with streptavidin,resulting in attachment. Alternatively, O may be synthesized on thesurface using techniques such as photopolymerization andphotolithography. Additional methods of attaching O to a surface andmethods of synthesizing O on surfaces are well known in the art, i.e.VLSIPS technology from Affymetrix (e.g., see U.S. Pat. No. 6,566,495,and Rockett and Dix, “DNA arrays: technology, options and toxicologicalapplications,” Xenobiotica 30(2):155-177, all of which are herebyincorporated by reference in their entirety).

(c) Use of Biosensors with No Detection Means

Alternatively, in certain embodiments it is contemplated that themolecular biosensor may not include a detections means. By way ofexample, when the molecular biosensor is a bivalent antibody construct,the bivalent antibody construct may not have labels for detection. It isenvisioned that these alternative bivalent constructs may be used muchlike antibodies to detect molecules, bind molecules, purify molecules(as in a column or pull-down type of procedure), block molecularinteractions, facilitate or stabilize molecular interactions, or conferpassive immunity to an organism. It is further envisioned that thebivalent antibody construct can be used for therapeutic purposes. Thisinvention enables the skilled artisan to build several combinations ofantibodies that recognize any repeating epitope from any number ofmolecules into a bivalent, trivalent, or other multivalent aptamerconstruct to pull together those disparate molecules to test the effector to produce a desired therapeutic outcome.

(d) Kits

In another embodiment, the invention is directed to a kit comprising afirst epitope binding agent, to which is attached a first label, and asecond epitope binding agent, to which is attached a second label,wherein (a) when the first epitope binding agent and the second epitopebinding agent bind to a repeating epitope of the polypeptide, (b) thefirst label and the second label interact to produce a detectablesignal. In a preferred embodiment the epitope-binding agent is anantibody construct, which comprises an antibody, a label and a signalingoligo. However, the epitope-binding agent may be an antibody fragment,an aptamer, or a peptide. The kit is useful in the detection of targetscomprising a repeating epitope, and as such, may be used in research ormedical/veterinary diagnostics applications. In particular, the kit maybe used to detect microbial organisms, such as bacteria, viruses, andfungi.

(e) Diagnostics

In yet another embodiment, the invention is directed to a method ofdiagnosing a disease comprising the steps of (a) obtaining a sample froma patient, (b) contacting the sample with a first epitope binding agentconstruct and a second epitope binding agent construct, and (c)detecting the presence of a polypeptide, microbial organism, ormacromolecular complex in the sample using a detection method, whereinthe presence of the polypeptide, microbial organism, or macromolecularcomplex in the sample indicates whether a disease is present in thepatient. In a one embodiment, (a) the first epitope binding agentconstruct is a first antibody to which a first label and a firstsignaling oligo are attached, (b) the second epitope binding agentconstruct is a second antibody to which a second label and a secondsignaling oligo, which is complementary to the first signaling oligo,are attached, and (c) the detection method is a fluorescence detectionmethod, wherein, (d) when the first antibody binds to the polypeptideand the second antibody binds to the polypeptide, (e) the firstsignaling oligo and the second signaling oligo associate with eachother, and (f) the first label is brought into proximity to the secondlabel such that a change in fluorescence occurs.

In another embodiment, (a) the first epitope binding agent construct isa first peptide to which a first label and a first signaling oligo areattached, (b) the second epitope binding agent construct is a secondpeptide to which a second label and a second signaling oligo, which iscomplementary to the first signaling oligo, are attached, and (c) thedetection method is a fluorescence detection method, wherein, (d) whenthe first peptide binds to the polypeptide and the second peptide bindsto the polypeptide, (e) the first signaling oligo and the secondsignaling oligo associate with each other, and (f) the first label isbrought into proximity to the second label such that a change influorescence occurs.

In other embodiments, the first epitope binding agent and the secondepitope-binding agents are different types of epitope binding agents(i.e. an antibody and a peptide, an aptamer and an antibody, etc.).Preferred samples include blood, urine, ascites, cells and tissuesamples/biopsies. Preferred patients include humans, farm animals andcompanion animals.

In yet another embodiment, the invention is directed to a method ofscreening a sample for targets comprising the steps of (a) contacting asample with a first epitope binding agent construct and a second epitopebinding agent construct, and (b) detecting the presence of a target inthe sample using a detection method. Preferred targets include apolypeptide, which comprises a repeating epitope, and a microbialorganism. In one embodiment, (a) the first epitope binding agent is afirst antibody to which a first label and a first signaling oligo areattached, (b) the second epitope binding agent is a second antibody towhich a second label and a second signaling oligo, which iscomplementary to the first signaling oligo, are attached, and (c) thedetection method is a fluorescence detection method, wherein, (d) whenthe first antibody binds to the polypeptide and the second antibodybinds to the polypeptide, (e) the first signaling oligo and the secondsignaling oligo associate with each other, and (f) the first label isbrought into proximity to the second label such that a change influorescence occurs.

In another embodiment, (a) the first epitope binding agent is a firstpeptide to which a first label and a first signaling oligo are attached,(b) the second epitope binding agent is a second peptide to which asecond label and a second signaling oligo, which is complementary to thefirst signaling oligo, are attached, and (c) the detection method is afluorescence detection method, wherein, (d) when the first peptide bindsto the polypeptide and the second peptide binds to the polypeptide, (e)the first signaling oligo and the second signaling oligo associate witheach other, and (f) the first label is brought into proximity to thesecond label such that a change in fluorescence occurs.

In other embodiments, the first epitope binding agent and the secondepitope-binding agents are different types of epitope binding agents(i.e. an antibody and a peptide, an aptamer and an antibody, etc.).

Definitions

The term “antibody” generally means a polypeptide or protein thatrecognizes and can bind to an epitope of an antigen. An antibody, asused herein, may be a complete antibody as understood in the art, i.e.,consisting of two heavy chains and two light chains, or be selected froma group comprising polyclonal antibodies, ascites, Fab fragments, Fab′fragments, monoclonal antibodies, humanized antibodies, and a peptidecomprising a hypervariable region of an antibody.

The term “aptamer” refers to a polynucleotide, generally a RNA or a DNAthat has a useful biological activity in terms of biochemical activity,molecular recognition or binding attributes. Usually, an aptamer has amolecular activity such as binding to a target at a specific epitope(region). It is generally accepted that an aptamer, which is specific inits binding to any polypeptide, may be synthesized and/or identified byin vitro evolution methods.

As used herein, “detection method” means any of several methods known inthe art to detect a molecular interaction event. The phrase “detectablesignal”, as used herein, is essentially equivalent to “detectionmethod.” Detection methods include detecting changes in mass (e.g.,plasmin resonance), changes in fluorescence (e.g., FRET, FCCS,fluorescence quenching or increasing fluorescence, fluorescencepolarization, flow cytometry), enzymatic activity (e.g., depletion ofsubstrate or formation of a product, such as a detectable dye—NBT-BCIPsystem of alkaline phosphatase is an example), changes inchemiluminescence or scintillation (e.g., scintillation proximity assay,luminescence resonance energy transfer, bioluminescence resonance energytransfer and the like), and ground-state complex formation, excimerformation, colorimetric substance detection, phosphorescence,electro-chemical changes, and redox potential changes.

The term “epitope” refers generally to a particular region of a target.Examples include an antigen, a hapten, a molecule, a polymer, a prion, amicrobe, a cell, a peptide, polypeptide, protein, a ligand, a receptor,or macromolecular complex. An epitope may consist of a small peptidederived from a larger polypeptide. An epitope may be a two orthree-dimensional surface or surface feature of a polypeptide, proteinor macromolecular complex that comprises several non-contiguous peptidestretches or amino acid groups.

The term “epitope binding agent” refers to a substance that is capableof binding to a specific epitope of an antigen, a polypeptide, a proteinor a macromolecular complex. Non-limiting examples of epitope bindingagents may include aptamers, double-stranded DNA sequence, ligands andfragments of ligands, receptors and fragments of receptors, antibodiesand fragments of antibodies, polynucleotides, coenzymes, coregulators,allosteric molecules, and ions.

The term “epitope binding agent construct” refers to a construct thatcontains an epitope-binding agent and can serve in a “molecularbiosensor” with another molecular biosensor. Preferably, an epitopebinding agent construct also contains a “linker,” and a “signalingoligo”. Epitope binding agent constructs can be used to initiate theaptamer selection methods of the invention. A first epitope bindingagent construct and a second epitope binding agent construct may bejoined together by a “linker” to form a “bivalent epitope binding agentconstruct.” An epitope binding agent construct can also be referred toas a molecular recognition construct. An aptamer construct is a specialkind of epitope binding agent construct wherein the epitope bindingagent is an aptamer.

The term “label”, as used herein, refers to any substance attachable toa polynucleotide, polypeptide, aptamer, nucleic acid component, or othersubstrate material, in which the substance is detectable by a detectionmethod. Non-limiting examples of labels applicable to this inventioninclude but are not limited to luminescent molecules, chemiluminescentmolecules, fluorochromes, fluorescent quenching agents, coloredmolecules, radioisotopes, scintillants, massive labels (for detectionvia mass changes), biotin, avidin, streptavidin, protein A, protein G,antibodies or fragments thereof, Grb2, polyhistidine, Ni2+, Flag tags,myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase,luciferase, electron donors/acceptors, acridinium esters, andcolorimetric substrates. The skilled artisan would readily recognizeother useful labels that are not mentioned above, which may be employedin the operation of the present invention.

As used herein, the term “macromolecular complex” refers to acomposition of matter comprising a macromolecule. Preferably, these arecomplexes of one or more macromolecules, such as polypeptides, lipids,carbohydrates, nucleic acids, natural or artificial polymers and thelike, in association with each other. The association may involvecovalent or non-covalent interactions between components of themacromolecular complex. Macromolecular complexes may be relativelysimple, such as a ligand bound polypeptide, relatively complex, such asa lipid raft, or very complex, such as a cell surface, virus, bacteria,spore and the like. Macromolecular complexes may be biological ornon-biological in nature.

The term “molecular biosensor” and “molecular beacon” are usedinterchangeably herein to refer to a construct comprised of at least twoepitope binding agent constructs. The molecular biosensor can be usedfor detecting or quantifying the presence of a target using achemical-based system for detecting or quantifying the presence of ananalyte, a prion, a protein, a nucleic acid, a lipid, a carbohydrate, abiomolecule, a macromolecular complex, a fungus, a microbial organism,or a macromolecular complex comprised of biomolecules using a measurableread-out system as the detection method.

The term “signaling oligo” means a short (generally 2 to 15 nucleotides,preferably 5 to 7 nucleotides in length) single-stranded polynucleotide.Signaling oligos are typically used in pairs comprising a firstsignaling oligo and a second signaling oligo. Preferably, the firstsignaling oligo sequence is complementary to the second signaling oligo.Preferably, the first signaling oligo and the second signaling oligo cannot form a stable association with each other through hydrogen bondingunless the first and second signaling oligos are brought into closeproximity to each other through the mediation of a third party agent.

EXAMPLES

The following examples illustrate various iterations of the invention.

Example 1 General Biosensor Design

The overall design of molecular biosensors is illustrated in FIG. 1.This concept is derived from molecular beacons for detecting DNA bindingproteins and from aptamer-based molecular beacons for detecting proteinsthat were previously developed [12-15]. A pair of antibodies recognizingnon-overlapping epitopes of the protein is labeled with a pair of shortcomplementary signaling oligonucleotides using a long flexible PEG-basedcrosslinker, respectively. Oligonucleotides are modified with a pair offluorophores that may function as a donor and an acceptor inFluorescence Resonance Energy Transfer (FRET) [16]. In the presence ofthe target protein both antibodies will bind to the target resulting ina great increase of the local concentration of signalingoligonucleotides. This in turn will lead to annealing of theoligonucleotides bringing the fluorophores to close proximity resultingin efficient FRET that can be used as a signal for target proteindetection. Recent quantitative analysis of binding properties of theligands containing long flexible linkers indicated that linkers with thelengths of tens of nanometers should be compatible with the designillustrated in FIG. 1A. Such long linkers should allow enough of roomand flexibility for effective formation of complexes containing twoantibodies and even very large protein.

Cardiac troponin I has been used as a target protein to demonstratefeasibility of the sensors depicted in FIG. 1A. When the labeledantibodies were mixed in the absence of cardiac troponin, no significantFRET signal was observed when fluorescence spectra of donor-labeledantibody in the absence and the presence of acceptor-labeled antibodywere compared (FIG. 1B). Upon addition of troponin, a large FRET signalwas observed illustrated by several fold increase of emission at 670 nmwith the excitation at 490 nm and quenching of donor emission at 520 nm.This signal allowed very sensitive (down to ˜40 μM troponin) detectionof the protein (FIG. 1C). Specificity of the sensor for cardiac troponinwas tested. No FRET signal was observed when nonspecific related controlprotein (porcine muscle troponin) was added to the sensor whereas arobust response with cardiac troponin was observed. Also, unlabeledcardiac troponin antibodies acted as competitors in the molecularbiosensor assay indicating that the signal generated by the molecularbiosensor in the presence of troponin was due to specific interactionsbetween labeled antibodies and the protein. High specificity ofmolecular biosensor is expected due to the necessary coincidence ofthree molecular events (the recognition of the target protein by each ofthe two antibodies and the association of the complementary signalingoligonucleotides) for generating signal in the presence of the target.In the absence of any one of these, the sensor will generate no signal.Generality of the sensor design has been confirmed by using the sameblueprint as used in the case of troponin for preparing a successfulsensor for other targets (C-reactive protein, insulin, C-peptide).

Example 2 Antibody-Based Biosensors for Detecting Bacteria

By analogy to the target-protein induced annealing of signalingoligonucleotides (FIG. 1), it was hypothesized that a similar effectcould be produced by binding of signaling oligonucleotide-labeledantibodies to the repeating epitopes on a surface of a bacterial cell(FIG. 2). Taking into account the dimensions of the bacterial cell(˜500-˜3000 nm) and the length of flexible linkers used to attach theoligonucleotides to the antibodies (10-50 nm), it was expected that evena moderate density of cell surface epitopes recognized by the antibodyshould be sufficient to bring the antibodies into the proximity thatwould allow annealing of the signaling oligonucleotides. This would inturn bring the donor and acceptor fluorochromes into the proximity toproduce FRET signal in the presence of target cells. It would seem thatsimilar to the molecular biosensor sensors for protein targets (FIG. 1)it would be necessary to have two antibodies (labeled with complementarysignaling oligonucleotides, respectively) to two distinct cell surfaceepitopes for the assay depicted in FIG. 2 to work. However, it washypothesized that it would be sufficient to use a mixture of the sameantibody labeled with two complementary signaling oligonucleotides,respectively. The reasons for that are illustrated in FIG. 3A.Complementary oligonucleotides provide additional favorable bindingenergy when the antibodies labeled with complementary oligonucleotidesbind next to each other. This energy is absent when the two neighboringantibodies are labeled with the same signaling oligonucleotide (anarrangement that does not generate FRET signal). Thus, when a mixture ofthe same antibody labeled with two complementary signalingoligonucleotides is used, the design has a built-in selection processwhereby the antibodies capable of producing FRET signal will bepreferentially bound near each other. The preliminary data describedbelow has fully validated the design illustrated in FIG. 2.

Example 3 E. coli Biosensors

To obtain proof-of-principle evidence validating the design illustratedin FIGS. 2 and 3A, a series of experiments was performed using E. coli0157:H7 as detection targets. Affinity-purified goat polyclonalantibodies specific for E. coli 0157:H7 were obtained from KPL. Twosamples of signaling oligonucleotide-labeled antibodies were preparedfor each of the above antibodies. One was modified (via a long flexiblelinker) with a 5′-fluorescein-GCTCAT and the other was modified with aCy5-modified complementary signaling oligonucleotide (5′-Cy5-ATGAGC).All fluorescence measurements were done using Analyst AD fluorescenceplate reader (Molecular Devices, Sunnyvale, Calif.).

When a mixture of E. coli 0157:H7 labeled antibodies was incubated withincreasing amounts of E. coli 0157:H7 cells, a large dose-dependent FRETsignal (emission at 670 nm with the excitation at 488 nm) was observed(FIG. 3B). In contrast, no FRET signal was recorded when the sameamounts of E. coli K12 cells were used demonstrating specificity of thesensor. FIG. 4 shows the results of the three independent repeats of theassay performed at a wide range of target cells amounts. Few thousandsof target cells were easily detectable using these assay conditions withhigh reproducibility (mean CV was 1.7%; range: 0.7-3.9%). No signal withnegative control (E. coli K12) has been detected at any amount of thecells tested. At very high amounts of the target cells, the FRET signalstarted to decrease most likely due to the decrease in the averageamount of antibodies bound to each cell due to the depletion of the freeantibody in the reaction mixture. Data shown in FIG. 3A and FIG. 4demonstrate the feasibility of the assay design illustrated in FIG. 2.

The assay design illustrated in FIG. 2 assumes that FRET signal isgenerated by annealing of complementary oligonucleotides attached to theantibodies co-binding to the same cell. The other possibility could bethat the FRET signal could be produced by annealing of signalingoligonucleotides attached to the antibodies bound to different cells(i.e. resulting in antibody-induced aggregation of the cells). Toinvestigate the nature of the FRET signal generated in the presence ofthe target cells, we have obtained confocal microscope images of E. coli0157:H7 cells incubated with the mixture of antibody labeled withfluorescein and Cy5-modified complementary oligonucleotides (FIG. 5;donor/acceptor) and the same cells incubated only with the antibodylabeled with fluorescein-modified signaling oligonucleotide that lacksthe ability of crosslinking via annealing of the complementaryoligonucleotides (FIG. 5; donor only). In both cases individual cellsand cells aggregates could be found. No increase in cell aggregation wasdetected in donor/acceptor sample compared to donor-only sample arguingagainst annealing of signaling oligonucleotides between different cellsas a mechanism for FRET signal. Furthermore, large FRET signal could beseen within the individual cells (FIG. 5, bottom FRET panel). Theseobservations indicate that the major route for the FRET signal producedin the presence of target cells is the process schematically illustratedin FIG. 2.

Example 4 Biosensor Response Time

One of the important advantages of the sensor design illustrated in FIG.2 is the uncomplicated rapid manner by which the assay can be performed.Thus, in the next experiment the response time of the sensor isdetermined. Various amounts of target cells were added to the mixture ofantibody labeled with fluorescein and Cy5-modified complementaryoligonucleotides and the FRET signal was monitored as a function ofincubation time (FIG. 6). Maximum FRET signal was obtained after ˜30 minincubation. After ˜5 min incubation >80% of the maximal FRET signal wasproduced (thus, if necessary, it would be possible to perform the assaywith 5 min incubation). These data confirm the rapid response time thebiosensor.

Example 5 Sensitivity

Detection sensitivity will be one of the important parameters that willdefine the practical utility of the biosensors. One way to optimizeassay sensitivity could be to find optimal concentrations of theantibodies. Reducing antibody concentrations could increase assaysensitivity since a bigger fraction of the total antibody concentrationmay be bound to cells at a given target cell density (provided that theantibody concentrations are not reduced below the Kd of antibody-antigencomplex). FIG. 7 shows the response of the biosensor at two (lower)antibody concentrations. As expected, lowering antibody concentrationsallowed detection of lower target cell amounts. It is estimated thatusing a 5/6.2 nM antibody mixture the sensitivity of detection was ˜150cells/20 ml assay.

Methods for bacteria detection often employ some form of sampleenrichment step to enhance detection of low cell amounts. A simple testwas performed to demonstrate that such step can be added to the assay.Two diluted samples of E. coli 0157:H7 were prepared. One (3000cells/ml; 60 cells/20 ml assay) was below the detection limit using asensor employing 20/25 nM mix of labeled antibodies (FIG. 8). The other(300,000 cells/ml; 6000 cells/20 ml assay) gave relatively low signalwith 20/25 nM mix of labeled antibodies (FIG. 8). Both samples were thenconcentrated ˜10 fold by a quick spin on a spin column with asemipermeable membrane and were re-measured. After the spin columntreatment, the sample that was previously undetectable produced easy tomeasure signal, whereas the signal for the more concentrated sample(that previously gave relatively low reading) was dramatically improved(FIG. 8).

Example 6 Biosensor to Salmonella

In order to demonstrate the generality of the sensor design illustratedin FIG. 2, we used the same procedure as used in the case of E. coliO157:H7 sensor to prepare a sensor for a different bacteria (Salmonellatyphimurium). FIG. 9 shows that as in the case of E. coli 0157:H7, alarge dose-dependent FRET signal was observed with Salmonella cells. Incontrast, very little FRET signal in the presence of E. coli K12 or E.coli O157:H7 was observed demonstrating the specificity of the sensorfor the target cells. As in the case of E. coli O157:H7 reproducibilitywas excellent (mean CV was 1.0%; range: 0.3-2.2%). FRET signal in thecase of Salmonella was smaller compared to E. coli O157:H7 most likelyreflecting differences in properties of corresponding antibodies (forexample, different surface density of the epitopes recognized by theantibodies). Nevertheless, the fact that the same design blueprintapplied to two different bacteria cells produced functional sensorsprovides a strong indication of the generality of the design illustratedin FIG. 2. Kinetics of Salmonella sensor response (FIG. 10) was similarto that observed for E. coli O157:H7 sensor.

Example 7 Preparation of Antibodies with Linkers

Preparation of the antibodies modified with short oligonucleotides vialong flexible linkers is an important step in preparing the biosensorsfor bacteria. A reliable procedure to label and purify the antibodieshas been developed. Briefly, the antibody is treated with Traut'sreagent to introduce free —SH groups. 5′-amino labeled oligonucleotideis reacted with NHS-maleimide bifunctional crosslinker producingmaleimide-containing oligonucleotide that is reacted with —SH containingantibody. A long flexible linker is introduced to the oligonucleotideeither during standard oligonucleotide synthesis [12] or can be a partof the bifunctional crosslinker. Labeled antibody is purified by FPLCsize exclusion chromatography.

Evaluating the pros and cons of various sensor design variants willinvolve performing the assays with a series of samples containing arange of dilution of each of the bacterial targets. Performance of thesensor will be evaluated by determining signal-to-background ratios,sensitivity and specificity of the detection. Sensitivity will bedefined as the lowest amount of the target producing a signal higherthan 3×standard deviation over the background measured with no target.Specificity will be evaluated by comparing the signals measured in thepresence of a given target with the signal measured at the same amountof unrelated bacteria.

Analysis of the properties of ligands with long flexible linkersindicated that the linkers with a length of up to 50 nm should produce afunctional sensor when used to attach the oligonucleotides to theantibody. However, these conclusions are based on experiments performedin solution using a simple model system. It is not clear if theseconclusions apply to our bacteria sensor design (FIG. 2) where theantibodies bind to a surface of micrometer-size cell. We will thuscompare the performance of the assays using antibodies labeled with 10,20, 30, 40 and 50 nm long linkers. Length of the linker can bemanipulated using constructs in which the flexible linker isincorporated during standard oligonucleotide synthesis [12]. Variouslengths of the linkers will be tried with all four bacterial targetssince it might be possible that the optimal length of the linker coulddepend on the surface density of the antigens recognized by theantibodies. For example, in preliminary experiments we have observedthat the FRET signal obtained in the case of Salmonella wassignificantly lower then in the case of E. coli O157:H7. It could bethat the FRET signal in the case of Salmonella could be improved byusing different length of the linker.

Example 8 Optimal Ratio of Oligonucleotide:Protein inAntibody-Oligonucleotide Conjugate

FIG. 2 schematically depicts antibodies labeled with one oligonucleotideper protein molecule. In reality, this does not need to be the case—itis unnecessary to prepare a homogenous preparation of such labeledantibodies. Degree of labeling of the antibodies can be modulated bychanging the concentrations of the reagents and/or the time of couplingreactions. The performance of the sensors with antibodies labeled witholigonucleotides to a different degree (from ˜1 to ˜5 oligonucleotidesper antibody) can be compared to find the optimal ratio.

Example 9 Optimal Length of the Signaling Oligonucleotide

The length of the oligonucleotides used to label the antibody will beimportant for optimal functioning of the sensors. To be more precise, itis not the length but the free energy change for their hybridization tothe complementary strand that is the parameter of interest. If thebinding affinity between the two complementary oligonucleotides used tolabel the antibody is too large, they will exhibit excessivetarget-independent association resulting in high background signal. Ifthe affinity is too small, the background binding will be small but thetarget-induced annealing of the oligonucleotides will be reducedresulting in suboptimal FRET signal in the presence of target cells. Inpreliminary experiments, oligonucleotides with hybridization ΔG at 0.1 MNaCl of ˜7.5 kcal/mole were used. While these oligonucleotides workedfine it is not known if they were optimal. The performance of thesensors utilizing the oligonucleotides with hybridization AG's in therange from 6 to 9 kcal/mole will therefore be tested to determine theoptimal AG value for high signal in the presence of the target cells andlow background in the absence of target.

Example 10 Dry-Chemistry Approach

To assure long-term stability, oligonucleotide-labeled antibodies havebeen stored frozen at −80°. When stored at 4°, they retain theirfunctionality for at least 2 weeks. These observations demonstrate thatthe stability of the main component of the sensors is similar to anyother immunological assay and is thus compatible with practicalapplications of the sensors. Homogenous assays in microplates can begreatly simplified by applying a dry-chemistry approach [17, 18]. Inthis approach the assay mixture solution is dried into the wells of themicroplate. If the assay reagents are compatible with this procedure,the assay is simplified since it requires only a simple addition of thesample solution to a microplate well with the dried assay components.The shelf life of the assay can be increased and the storagerequirements simplified when dry-chemistry approach is applied.

Thus, the performance of the sensors will be tested using adry-chemistry approach. The sensor mixtures (5 ml labeled antibodysolution in a buffer containing 5% sorbitol which was shown tofacilitate retention of functional properties of assay reagents afterdrying [18]) will be evaporated in the wells of 384-well microplateovernight in a desiccator over silica gel [17, 18]. The sensitivity ofthe assays for the four bacterial targets will be compared using thedry-chemistry assay with the assays performed using the “normal” wetchemistry approach. If no significant decrease of assay sensitivity dueto drying and dissolution of the assay components are observed, thestability of the assay in dry-chemistry format will be further tested.The plates with dry assay components sealed with a plate sealing filmwill be stored at room temperature or at 4° for an extended period oftime (24 weeks). The performance of the stored dry-chemistry assays willbe evaluated in weekly intervals to determine the effect of time ofstorage and storage temperature on the assay performance.

Example 11 Improving Sensitivity

Sensitivity of target cell detection will define where and how thesensors can be ultimately used. Current sensitivity of the sensors asdetermined in the preliminary experiments is equal or better to ELISAassays for bacteria detection [2]. Thus, even before any optimization ofthese sensors has been performed, they appear to be a superioralternative to standard ELISA assays since they offer the samesensitivity but the results can be obtained in a fraction of timerequired to perform ELISA and they are vastly simpler to perform. Forthe detection of the amounts of cells below the current sensitivitylimit, the sample potentially could be enriched using semipermeablemembrane concentration step (as illustrated by preliminary dataillustrated by FIG. 8) or an cell culture enrichment step could beperformed. Nevertheless, the more sensitive the sensors will be, themore applications they could potentially find. The following ideas forimproving assay sensitivity may thus be tested:

(i) Alternative FRET Probes. One relatively straightforward approach toimprove sensor sensitivity will be to use better fluorescence probes(probes with increased brightness). It will be tested if replacing thefluorescein-Cy5 donor-acceptor pair used in all preliminary experimentswith a brighter and more photostable pair of probes (Alexa488/Alexa647or Alexa546/Alexa647) can produce an increase in sensitivity.

(ii) Time-Resolved FRET (LRET). Another potential approach to increaseFRET based assay sensitivity is to increase signal-to-background ratioof FRET signal. Majority of background signal in FRET-based assaysoriginates from the spillover of the donor emission to the acceptordetection channel and from direct excitation of the acceptor at donorexcitation wavelength. These two sources of the elevated background (aswell as the background due to light scattering) can be effectivelyremoved by employing Luminescence Energy Transfer (LRET) usinglanthanide chelates as donor labels [19-23]. These labels exhibit verylong (hundreds of microseconds) fluorescence lifetimes allowing theemployment of pulsed excitation and delayed gated emission measurementsthat have been shown to dramatically reduce background signals inFRET-based homogenous assays [19, 22]. LRET is also very effective inremoving the background due to light scattering or due to fluorescentimpurities in the sample. This could be beneficial when turbid orautofluorescent samples need to be analyzed. For each of the fourbacterial targets, antibodies labeled with complementaryoligonucleotides modified with europium chelate and with Cy5,respectively, will be prepared. The performance of the sensors utilizingLRET-based detection will be compared with the sensors utilizingstandard FRET detection.

(iii) Restriction-Enzyme Based Signal Amplification. For the detectionof protein targets in solution using the molecular biosensor assay(FIG. 1) a restriction-enzyme digestion based signal amplificationmethodology (FIG. 11) has recently been developed that could allow ˜100fold signal amplification compatible with the homogenous nature of theassay. This amplification approach is based on target-dependentrestriction enzyme digestion of fluorescent DNA construct (S3, FIG. 11)whose fluorescence is dramatically increased upon its cleavage. Signalamplification is derived from multiple rounds of cleavage that a singletarget-antibody complex can catalyze. S3 component contains a sequencerecognized by a restriction enzyme. Hinc II sequence will be used butany restriction enzyme that cleaves ds DNA but is inactive on ss DNA(like Hinc II) could be used. S3 also contains the probes attached totwo complementary oligonucleotides that are in turn attached to S3 viaflexible linkers. When S3 oligonucleotide is intact, the complementaryoligonucleotides will be annealed (generating proximity-dependent signalsuch as, for example, FRET) due to the high local concentrationresulting from their attachment to S3. Oligonucleotides used in S1 andS2 are designed to be sufficiently short that on their own they cannotanneal to S3 in any significant manner. In the presence of the target,S1 and S2 in a complex with the target will bind to S3 because thecomplex becomes effectively a bivalent binder for S3 that will have muchincreased S3 binding affinity compared to free S1 or S2. Theoligonucleotides of S1 and S2 are designed to anneal to S3 such that agap between the two oligonucleotides hybridized to S3 is exactly at theposition where Hinc II would normally cleave the bottom strand of DNAduplex. Thus, when Hinc II is present in the sample, it will cleave S3only when it is annealed to both S1 and S2 (i.e. when the target ispresent). Upon cleavage of S3, the complex will dissociate (cleavage ofS3 will greatly decrease both the stability of the complex as well as itwill result in dissociation of the two signaling oligonucleotides whichin turn will eliminate the proximity-dependent signal). The complex of Twith S1 and S2 can now associate with another molecule of S3 and suchcleavage and dissociation cycle could be repeated many times producingsignal amplification. We propose that the same events as thoseillustrated in FIG. 11 for a protein target in solution could beproduced by the antibodies associating with the surface of the bacteriacell (as illustrated in FIG. 2). The applicability of the signalamplification scheme illustrated in FIG. 11 will be tested for detectingbacteria by preparing bacteria-specific antibodies labeled witholigonucleotides complementary to S3. A mixture of such antibodies willbe incubated with various amounts of target and negative control cellsin the presence of Hinc II followed by fluorescence intensitymeasurement. The performance of the assays employing this amplificationscheme will be compared to assays employing standard FRET detection.

Example 12 Solid-Surface Based Variant of the Biosensor

The goal of this example will be to develop an alternative design of thesensor illustrated in FIG. 12. While implementing this sensor designwill require a more sophisticated instrumentation, its potentialadvantages include an increase in sensitivity and the possibility formultiplexed simultaneous detection of several targets. In this design,an antibody modified with a single short oligonucleotide via a longflexible linker will be used (FIG. 12). The second oligonucleotidecontaining a segment(s) complementary to this oligonucleotide will beimmobilized on a solid surface. The oligonucleotide that will be used tolabel the antibody will be designed to provide some affinity for bindingto the immobilized complementary oligonucleotide but this affinity willbe set (affinity of the interaction between two oligonucleotides can beset at any desired level using length and the sequence ofoligonucleotides as a variable) to be low enough that at nanomolarconcentrations of the antibody very little association of the freeantibodies with the immobilized oligonucleotide will be observed (forexample, a KD in mM range would fulfill this design requirement). Wehypothesize that when the target cells are added, the antibodies boundto the surface of the cell will in effect create a multivalent particlecapable of multivalent interactions with the immobilizedoligonucleotides (FIG. 12). Due to the multivalent character of theseinteractions, the target cells should bind to the solid surface withvery high affinity. We suggest that this binding of the target cells tothe solid surface induced by the association of the labeled antibodywith the cell surface antigens can be utilized for effective detectionof the target cells. FIG. 13 illustrates a feasibility experimentdemonstrating detection of target bacteria with the solid-surface assayaccording to a design illustrated in FIG. 12.

Since the antibodies are labeled with fluorescence probes, anyfluorescence detection specific to surface-bound fluorescent probescould be potentially used for the readout of the signal. The performanceof two variants of this alternative assay design will be investigated.In the first variant, oligonucleotide complementary to theantibody-bound oligonucleotide will be immobilized in the well of a96-well microplate. The sample will be incubated witholigonucleotide-labeled antibody followed by washing the wells with thebuffer and readout of fluorescence remaining in the well. Second variantwill involve a real-time readout of the association of the targetcell-antibody complex with the solid surface using Total InternalReflection Fluorescence (TIRF). This mode of signal readout will involvemore technically complex instrumentation but will likely be moresensitive and faster. Additionally, it would be possible to immobilizeoligonucleotides of unique sequences at different areas (spots) of TIRFslide. This would allow a simultaneous multiplexed detection of severaltarget cells by using antibodies specific to each of the target celllabeled with a unique sequence oligonucleotide complementary tooligonucleotide immobilized at a unique spot of the slide.

Most of the parameters of the optimal homogenous assay to be determinedin above will be also applicable to the solid surface-based assay.However, the optimal length of the oligonucleotides attached to theantibody in the case of the assay illustrated in FIG. 12 is likely to bedifferent than in the homogenous assay (FIG. 2). It is likely that loweraffinity oligonucleotides may be required for optimal performance in thecase of solid-surface based assay. The performance of the solid-surfacevariant of the assay (FIG. 12) will therefore be tested utilizing theoligonucleotides with hybridization ΔG's in the range from 5 to 8kcal/mole. In these experiments, oligonucleotides complementary to theantibody-bound oligonucleotides will be immobilized in the well of a96-well microplate. This version of the solid-surface based assay isrelatively easy to set up and perform and the conclusions regarding theoptimal length of the complementary oligonucleotides obtained with thisassay variant should be also applicable to the real-time TIRF-basedassay. Streptavidin-coated plates and biotinylated oligonucleotide willbe used to immobilize the oligonucleotides on a surface of themicroplate well. For each oligonucleotide length tested, various amountsof target cells and in parallel, negative control cells, will beincubated in the wells of the microplate coated with the correspondingcomplementary oligonucleotide. The wells will be washed three times withthe buffer and fluorescence remaining in each well will be measured onAnalyst AD microplate reader (Molecular Devices, Sunnyvale, Calif.).Sensitivity of detection and signal-to-background ratio for eacholigonucleotide length tested will be used to determine the optimallength of the oligonucleotide.

Immobilized oligonucleotides containing multiple repeats of the sequencecomplementary to the oligonucleotide used to label the antibody willalso be tested (as opposed to a single copy of such sequence as depictedin FIG. 12). The affinity of the target cell coated witholigonucleotide-labeled antibodies for binding to the solid-surfacecontaining oligonucleotides with multiple repeats of the complementarysequence may be greatly improved which would be beneficial for theperformance of solid-surface based assay.

Example 13 TIRF Detection

TIRF (Total Internal Reflection Fluorescence) [31-34] uses evanescentwave generated near the surface of the glass slide by a light beamreflected from the slide to limit excitation to the molecules bound toslide surface. Limiting the excitation only to molecules bound to theslide surface allows real-time monitoring of the interactions of labeledmolecules with the slide surface since the fluorescent molecules in thebulk solution are not excited and do not contribute to the signal. Also,limiting excitation to a very thin (few hundred nanometers typically)layer of the sample reduces background, increasing the sensitivity ofdetection. This allows the detection of fluorescence even from singlemolecules [31, 34]. TIRF can be thus adopted for real-time detection oftarget cell induced association of the antibody-coated cell to the slidesurface coated with the oligonucleotides complementary to theoligonucleotide used to label the antibody. Real-time detectioncapability will allow rapid readout time and the outstanding signaldetection sensitivity of TIRF can produce significant sensitivity gains.FIG. 14 illustrates the feasibility of experiments demonstratingdetection of target bacteria with the solid-surface assay implementingTIRF detection.

Additionally, TIRF-based detection offers a unique potential ofdeveloping a multiplexed assay that will allow simultaneous detection ofmultiple target cells. To test this, four oligonucleotides of uniquesequence will be immobilized to four distinct areas (“spots”) on theTIRF slide. The antibodies for the four targets will be labeled, eachwith a unique sequence oligonucleotide complementary to theoligonucleotide immobilized in one of the spots. When a samplecontaining the target cells mixed with the oligonucleotide-labeledantibodies for the four targets will be introduced to the TIRF slide,the presence of a specific target cell will be detected by theappearance of the fluorescence signal at the spot containing animmobilized oligonucleotide of the sequence complementary to theoligonucleotide used to label the antibody for this target. To test thismultiplexed TIRF-based assay, samples containing only one of the targetsat a time will be tested to verify that fluorescence signal will bedetected only at a correct spot. Samples containing mixtures of targetcells at various proportions will then be added to test the ability ofthe multiplexed assay to correctly report the composition of thesecomplex samples.

Example 14 Biosensor Design for Detecting Viruses

As in the case of pathogenic bacteria, rapid detection andidentification of viruses is an essential element of infectious diseasediagnosis, treatment and prevention. For example, rapid detection ofavian flu infection in poultry is critical to controlling outbreaks.Virus particles, while much smaller then bacterial cells (for example,influenza virus is 80-120 nm in diameter), should be still large enoughto produce FRET signal resulting from co-binding of many labeledantibodies to surface epitopes of the virus according to scheme in FIG.2. Influenza A and B viruses will be used as model targets toinvestigate the applicability of our sensors for detecting viruses. Thegamma-radiation inactivated viruses and the virus-specific antibodieswill be purchased from BiosPacific (Emeryville, Calif.).

If the results of experiments described above show the generalfeasibility of applying sensor design illustrated in FIG. 2 for virusdetection, the performance of the solid-surface based assay will betested for detecting viruses. Both the microplate-based assay andTIRF-based assay will be evaluated. These data will be used to determinesensitivity, specificity and reproducibility (CV's) of the optimizedassays. These parameters will be used to evaluate the feasibility ofemploying the solid-surface based assays for detecting viruses.

Example 15 Detection of a Protein Comprising a Repeating Epitope

An anti-human Ferritin antibody was converted into a molecular biosensorpair (anti-human Ferritin-A2-AA2-FAM and anti-humanFerritin-A2-AM-Oyster). These two molecular pincers were mixed as a 2×assay mixture (40 nM of each pincer, calculated as oligo concentration)in TBS with 0.1 mg/ml BSA. The purified human Ferrintin protein standardwas diluted in 10 μl TBS with 0.1 mg/ml BSA range from 200 nM to OnM. 10μl of the 2× assay solution was added to 10 μl of each sample. The platewas incubated at RT for 40 min and the FRET (485 nm/665 nm) and FAM (485nm/535 nm) was recorded on a TECAN plate reader. The relativefluorescence fold change (RF) was calculated from raw fluorescencevalues using the following formula where RF=Relative fluorescence foldchange, FRET_(S)=FRET signal of the sample, FRET₀=FRET signal of theblack, FRET_(B)=background FRET signal (buffer alone), FAM_(S)=FAMsignal of the sample, FAM₀=FAM signal of the blank, FAM_(B)=backgroundFAM signal (buffer alone).RF=(FRET _(S) −FRET _(S))(FAM ₀ −FAM _(S))/(FRET ₀ −FRET _(S))(FAM _(S)−FAM _(B))

What is claimed is:
 1. A method for detecting a target comprising atleast one repeating epitope, the method comprising contacting a samplecomprising the target with a molecular biosensor, the biosensorcomprising:R²⁴—R²⁵—R²⁶—R²⁷,R²⁸—R²⁹—R³⁰—R³¹, andO wherein: R²⁴ is a peptide epitope binding agent that binds to arepeating epitope on a target antibody; R²⁵ is a flexible linkerattaching R²⁴ to R²⁶; R²⁶ and R³⁰ are a pair of nucleotide sequencesthat are not complementary to each other, but are complementary to twodistinct regions on O; R²⁷ and R³¹ together comprise a detection meanssuch that when R²⁶ and R³⁰ associate with O a detectable signal isproduced; R²⁸ is a peptide epitope binding agent that binds to the samerepeating epitope on the target antibody as R²⁴; R²⁹ is a flexiblelinker attaching R²⁸ to R³⁰; and O is a nucleotide sequence comprising afirst region that is complementary to R²⁶, and a second region that iscomplementary to R³⁰, and detecting the signal produced by theassociation of R²⁶ with O and R³⁰ with O, wherein the signal indicatesthe presence of the target antibody.
 2. The method of claim 1, whereinthe detection means is selected from the group consisting of FRET,fluorescence cross-correlation spectroscopy, fluorescence quenching,fluorescence polarization, flow cytometry, scintillation proximity,luminescense resonance energy transfer, direct quenching, ground-statecomplex formation, chemiluminescence energy transfer, bioluminescenceresonance energy transfer, excimer formation, colorimetric substratesdetection, phosphorescence, electro-chemical changes, and redoxpotential changes.
 3. The method of claim 1, wherein 80% of the maximumsignal may be detected at 5 minutes.
 4. The method of claim 1, wherein Ocomprises formula (IV):R³²—R³³—R³⁴—R³⁵—R³⁶  (IV) wherein: R³², R³⁴, and R³⁶ are nucleotidesequences not complementary to any of R²⁶, R³⁰, R³³, or R³⁵; R³³ is anucleotide sequence complementary to R²⁶; R³⁵ is a nucleotide sequencethat is complementary to R³⁰; R³³ is not adjacent to R³⁵; and whereinR³², R³⁴, and R³⁶ are from 2 to 20 nucleotides in length; and R³³ andR³⁵ comprise a length such that the free energy of association betweenR³³ and R²⁶ and R³⁵ and R³⁰ is from −5 to −12 kcal/mole at a temperaturefrom 21° C. to 40° C. and at a salt concentration from 1 mM to 100 mM.5. The method of claim 1, wherein R²⁵ and R²⁹ are from 50 to 250angstroms in length and are independently selected from the groupconsisting of a heterobifunctional chemical linker, a homobifunctionalchemical linker, polyethylene glycol, and nucleic acid.
 6. The method ofclaim 1, wherein R²⁶ and R³⁰ are from 2 to 20 nucleotides in length. 7.A method for detecting a target in a sample, the method comprising: (a)contacting a surface comprising an immobilized oligonucleotide construct0, with two epitope binding agents constructs, and a sample, the epitopebinding agents constructs comprising:R²⁴—R²⁵—R²⁶—R²⁷;R²⁸—R²⁹—R³⁰—R³¹; wherein: R²⁴ is a peptide epitope binding agent thatbinds to a repeating epitope on a target antibody; R²⁵ is a flexiblelinker attaching R²⁴ to R²⁶; R²⁶ and R³⁰ are a pair of nucleotidesequences that are not complementary to each other, but arecomplementary to two distinct regions on O; R²⁷ and R³¹ are labels thattogether comprise a detection means such that when R²⁶ and R³⁰ associatewith O a detectable signal is produced; R²⁸ is a peptide epitope bindingagent that binds to the same repeating epitope on the target antibody asR²⁴; R²⁹ is a flexible linker attaching R²⁸ to R³⁰; and O is anucleotide sequence comprising a first region that is complementary toR²⁶, and a second region that is complementary to R³⁰, wherein O isimmobilized to the surface irrespective of the association between R²⁶and O or R³⁰ and O; and (b) detecting whether R²⁶ and R³⁰ bind to O,wherein the binding indicates that the target antibody is present in thesample.